Human papillomavirus (HPV) is the most common sexually-transmitted infection, and the cause of nearly all cervical and anogenital, and over half of oral cancers. Current HPV treatment is by lesion removal or through immunological intervention (imiquimod as an immune stimulant, or the HPV vaccines to prevent infection of the most common HPVs). While antiviral agents have been developed against many types of viruses, to date no true antivirals are available against HPV. With the heavy reliance on the immune system for HPV treatment/prevention, HPV infections and cancers remain a major problem for HIV/AIDS patients, even after HAART treatment. A true HPV antiviral that acts directly against HPV would be an important weapon against HPV infections and cancers. Recent successes of small molecule inhibitors that interfere with the herpesvirus primase- helicase interaction justify using such an approach against HPV. We have identified an interaction between the HPV DNA replication helicase, E1, and human Topoisomerase I that is vital for HPV genome duplication. Aim 1 will evaluate a panel of E1 mutations predicted to disrupt the interaction with Topoisomerase to more fully define this interaction. In Aim 2 peptide binding will be used to further define the interaction domain, and our E1-Topoisomerase interaction assays will be used to screen panels of peptides and small molecules to identify first-generation inhibitors of this interaction. In Aim 3 the structure of this interaction will be evaluted using predictive software, NMR, X-ray crystallography, and SAXS. Each Aim can be achieved independently, yet information from each synergizes and enhances the others. Multiple achieveable approaches in each Aim allows for attaining the goals without requiring success of all approaches in each Aim. Results will provide chemical and structural information that will be used in developing second-generation inhibitors that will be investigated as potential antiviral therapeutics against papillomaviruses.